The overall objective is to gain a more complete understanding of the structure of apolipoprotein (apo) E, especially as it relates to its anti-atherogenic properties, including the ability of the protein to bind to lipids, to heparin and other glycosaminoglycans (GAG), and to members of the low density lipoprotein receptor (LDLR) family. A range of engineered apo E molecules expressed in E. coli is being used to address 3 specific aims. 1) To understand the molecular features that control the binding of apoE and its common isoforms to lipid and lipoprotein particles of different sizes, the interactions of engineered forms of the protein with the surface of phospholipid-containing particles will be characterized using both in vitro and in vivo approaches. The hypothesis being tested is that formation of amphipathic a-helices with appropriate properties in the C-terminal domain of apoE controls lipid binding. 2) To characterize the lipid efflux and nascent apoE-HDL particle formation that occurs when apoE is a ligand for the ATP binding cassette transporter A1 (ABCA1) in neuronal cells. The hypothesis being tested is that certain properties of C-terminal a-helices in apoE control the sizes and compositions of the HDL particles released into the extracellular medium. 3) To understand how the receptor binding domain of apoE modulates its binding to different sulfated proteoglycans and GAG as compared to members of the LDLR family, the affinities and kinetics of interaction will be determined using surface plasmon resonance and cell-based assays. The hypothesis being tested is that high affinity interaction with different types of receptor requires a specific basic microenvironment in the apoE molecule. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apoE structure and polymorphism affect the functional properties of the protein with respect to both lipid transport and cell signalling events. The design of apoE- mimetic molecules and of ways to control the aberrant behavior of certain isoforms of apoE will be facilitated by this understanding. Relevance In the human population, apoE is expressed in 3 common forms that function differently in regulating lipid transport in vivo. As a result, these mutations in the apoE molecule can increase the risk for development of both cardiovascular disease and Alzheimer's disease. This project will provide more insights into the molecular mechanisms underlying these pathological effects.